This invention relates to the use of compounds capable of inhibiting the activity of a proton-gated cation channel in the treatment or alleviation of diseases or disorders associated with, or mediated by a drop in extracellular pH.
For almost 20 years, proton-gated cation channels have been known to exist in membranes of sensory neurons, where they are assumed to participate in nociception. Recently, several Acid Sensing Ion Channels (ASIC) have been cloned, and while some of these like ASIC3 (DRASIC) and ASIC-xcex2 are selectively expressed in sensory ganglia and/or the spinal cord, others, like ASIC1 and ASIC2, are expressed also in the brain [see e.g. Waldmann R, Champigny G, Bassilana F, Heurteaux C, Lazdunski M: A proton-gated cation channel involved in acid-sensing; Nature 1997 386 (6621)173-177; Lingueglia E, De Weille J R, Bassilana F, Heurteaux C, Sakai H, Waldmann R, Lazdunski M: A modulatory subunit of acid sensing ion channels in brain and dorsal root ganglion cells; J. Biol. Chem. 1997 272 (47) 29778-29783; Bassilana F, Champigny G, Waldmann R, De Weille J R, Heurteaux C, Lazdunski M: The acid-sensitive ionic channel subunit ASIC and the mammalian degenerin MDEG form a heteromultimeric H+-gated Na+ channel with novel properties; J. Biol. Chem. 1997 272 (46) 28819-28822; Olson T H, Riedl M S, Vulchanova L, Ortiz-Gonzalez X R, Elde R: An acid sensing ion channel (ASIC) localizes to small primary afferent neurons in rats; NeuroReport 1998 9 (6) 1109-1113; Chen C -C, England S, Akopian A N, Wood J N: A sensory neuron-specific, proton-gated ion channel; Proc. Natl. Acad. Sci. USA 1998 95 (17) 10240-10245; and Coscoy S, de Weille J R, Lingueglia E, Lazdunski M: The pre-transmembrane 1 domain of acidxe2x80x94sensing ion channels participates in the ion pore; J. Biol. Chem. 1997 274 (15) 10129-101321].
The proton-gated channels cloned all belong to the amiloride-sensitive Na-channel/degenerin family of ion channels, and like for ionotropic purinergic receptors, the topology presumably is two transmembrane domains flanking a long extracellular loop.
Compounds inhibiting the proton-gated cation channels have been suggested useful for the treatment of pain.
WO 98/35034 discloses mammal neuronal acid sensing cationic channels, which are considered useful for screening for analgesic drugs and for the treatment of degeneration of periferic neurons
Since the demonstration that the level of extracellular glutamate in the brain is greatly elevated under ischaemia, much pharmaceutical research has been focused on design of glutamate receptor antagonists. There has, however, been discrepancy between in vitro and in vivo studies: especially with respect to the effect of antagonists of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. In vitro, neuro degeneration due to oxygen/glucose deprivation is blocked by the NMDA receptor antagonist MK-801, but not by xcex1-amino-3-hydoxy-5-methyl4-isoxazolepropionic acid (AMPA) receptor antagonists. Strikingly, the case is opposite in in vivo models of global cerebral ischaemia: AMPA receptor antagonists reduces infarct volume, while NMDA receptor antagonists are ineffective.
Isatin derivatives like those described herein, for use as AMPA antagonists have been disclosed in e.g. WO 94/26747, WO 96/08494 and WO 96/08495. However, the use of these isatin derivatives as ASIC antagonising compounds have never been disclosed.
The present work shows the presence of ASIC in cultured mouse cortical neurons, a preparation widely used for studying native ion channels and in vitro models of neuro degeneration after ischaemia.
The presence of ASIC in central neurons is of particular interest because tissue acidosis is a well established feature of cerebral ischaemia. While the acidic pH in general has been regarded as neuro-protective due to proton inhibition of NMDA receptors, it may also have adverse effects by reason of activation of acid sensing ion channels contributing to membrane depolarisation, subsequent Ca2+ accumulation and neuro-degeneration.
Therefore, in a first aspect, the invention relates to the use of a compound capable of inhibiting a mammalian proton-gated cation channel for the manufacture of a medicament for the treatment, prevention or alleviation of a disease, disorder or condition associated with, or mediated by a drop in extracellular pH.
In another aspect the invention provides a method for the treatment, prevention or alleviation of a disease, disorder or condition in a subject, including a human, which disease, disorder or condition is associated with, or mediated by a drop in extracellular pH, said method comprising administering to the subject a pharmaceutically effective amount of a compound capable of inhibiting a mammalian proton-gated cation channel.
Other objects of the invention will be apparent to the person skilled in the art from the following detailed description and the examples.
ASIC Antagonising Compounds
In its first aspect the invention relates to the use of compounds capable of inhibiting a mammalian proton-gated cation channels for the manufacture of medicaments for the treatment, prevention or alleviation of a disease, disorder or condition associated with, or mediated by a drop in extracellular pH.
In the context of this invention compounds capable of inhibiting a mammalian proton-gated cation channel is also designated ASIC antagonising compounds.
Method of Screening
The ASIC antagonising compounds of the invention may be identified using the following screening method, which method comprises the subsequent steps of
(i) subjecting a proton-gated cation channel containing cell to the action of protons by adjustment of the pH to an acidic level;
(ii) subjecting a proton-gated cation channel containing cell to the action of the chemical compound; and
(iii) monitoring the change in membrane potential or the current induced by protons on the proton-gated cation channel containing cell.
In a preferred embodiment, the proton-gated cation channel is ASIC1 (also known as human brain sodium channel channel 2 (hBNaC2)), ASIC1A, ASIC1B, ASIC2 (also known as human brain sodium channel channel 1 (hBNaC1), or MDEG1), ASIC2A, ASIC2B, ASIC3 (also known as DRASIC), or ASIC-xcex2.
The proton-gated cation channel may or may not be endogenous to the cell in question, i.e. be a channel naturally occurring in the cell.
Cells for use in the method of the invention, in which proton-gated cation channel are naturally present includes cortical neuronal cells, in particular mouse or rat cortical neuronal cells, and human embryonic kidney (HEK) cells, in particular HEK 293 cells.
Alternatively the proton-gated cation channel may be exogenous to the cell in question, and may in particular be introduced by recombinant DNA technology, such as transfection or infection. Such cells include Chinese hamster ovary (CHO) cells, Xenopus laevis oocytes, or any other cell lines capable to express proton-gated cation channels.
The proton-gated cation channel containing cells may be subjected to the action of protons by adjustment of the pH to an acidic level using any convenient acid or buffer, including organic acids such as formic acid, acetic acid, citric acid, ascorbic acid and lactic acid, and inorganic acids such as hydrochloric acid, hydrobromic acid and nitric acid, perchloric acid and phosphoric acid.
In the method of the invention, the current flux induced by protons over the membrane of the proton-gated cation channel containing cell may be monitored by patch clamp techniques.
Alternatively, the change in membrane potential induced by protons of the proton-gated cation channel containing cells may be monitored using fluorescence methods. When using fluorescence methods, the proton-gated cation channel containing cells are incubated with a membrane potential indicating agent, that allow for a determination of changes in the membrane potential of the cells, caused by the added protons. Such membrane potential indicating agents include fluorescent indicators, preferably DIBAC4(3), DiOC5(3), and DiOC2(3).
In a preferred embodiment, the change in membrane potential induced by protons on the proton-gated cation channel containing cells are monitored by spectroscopic methods, e.g. using a FLIPR assay (Fluorescence Image Plate Reader; available from Molecular Devices).
The ASIC antagonising compounds of the invention show activity in concentrations below 100 xcexcM, preferably below 10 xcexcM, more preferred below 1 xcexcm. In its most preferred embodiment the ASIC antagonising compounds show activity in low micromolar and the nanomolar range.
Preferred ASIC Antagonising Compounds
In a preferred embodiment the compound for use according to the present invention is an isatin derivative represented by the general Formula I 
or a pharmaceutically acceptable salt thereof, wherein
R1 represents hydrogen, alkyl, phenyl or benzyl; and
X represents oxygen, or a group of the formula NOR2 (i.e. an oxim), wherein R2 represents hydrogen, alkyl, acyl, phenyl or benzyl; and
Y represents a group of the formula Nxe2x80x94R4, wherein R4 represents hydrogen, hydroxy or alkyl; and
n is 0 or 1; and
R6 represents phenyl, naphthyl, thienyl, or pyridyl, all of which groups may be substituted one or more times with halogen, CF3, nitro, amino, cyano, hydroxy alkyl, alkoxy or phenyl, or a group of the formula xe2x80x94SO2NRxe2x80x2Rxe2x80x3, wherein NRxe2x80x2 and Rxe2x80x3 independently of each another represents hydrogen or alkyl; and
xe2x80x9cAxe2x80x9d represents a ring holding of from five to seven atoms, which ring is fused to the benzo-ring at the positions marked xe2x80x9caxe2x80x9d and xe2x80x9cbxe2x80x9d, and formed by one or the following bivalent radicals:
(a) xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94 (b),
(a) xe2x80x94NR12xe2x80x94CH2xe2x80x94CH2xe2x80x94 (b),
(a) xe2x80x94CH2xe2x80x94NR12xe2x80x94CH2xe2x80x94 (b),
(a) xe2x80x94CH2xe2x80x94CH2xe2x80x94NR12xe2x80x94 (b),
(a) xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94 (b),
(a) xe2x80x94NR12xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94 (b),
(a) xe2x80x94CH2xe2x80x94NR12xe2x80x94CH2xe2x80x94CH2xe2x80x94 (b),
(a) xe2x80x94CH2xe2x80x94CH2xe2x80x94NR12xe2x80x94CH2xe2x80x94 (b),
(a) xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94NR12xe2x80x94 (b),
(a) xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94 (b),
(a) xe2x80x94NR12xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94 (b),
(a) xe2x80x94CH2xe2x80x94NR12xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94 (b),
(a) xe2x80x94CH2xe2x80x94CH2xe2x80x94NR12xe2x80x94CH2xe2x80x94CH2xe2x80x94 (b),
(a) xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94NR12xe2x80x94CH2xe2x80x94 (b), or
(a) xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94NR12xe2x80x94 (b),
wherein R12 represents hydrogen, alkyl, alkyl substituted with hydroxy, alkoxy-alkyl, alkoxy-carbonyl-alkyl, alkyl-carbonyl-oxy-alkyl, cycloalkyl, cycloalkyl-alkyl, alkenyl, alkynyl, phenyl or benzyl, which phenyl or benzyl group is optionally substituted with halogen, CF3, nitro, amino, cyano, hydroxy alkyl, alkoxy, or a group of the formula xe2x80x94SO2NRxe2x80x2Rxe2x80x3, wherein Rxe2x80x2 and Rxe2x80x3 independently of each another represents hydrogen or alkyl.
In another preferred embodiment, the compound for use according to the present invention is represented by the general Formula II 
or a pharmaceutically acceptable salt thereof,
wherein R1, R6, R12, X, Y and n have the meanings set forth above.
In a third preferred embodiment, the compound for use according to the present invention is represented by the general Formula III 
or a pharmaceutically acceptable salt thereof,
wherein R1, R6, R12 and X have the meanings set forth above.
In a more preferred embodiment, the compound represented by Formula III is
8-ethyl-5-phenyl-6-7-8-9-tetrahydro-1H-pyrrolo-[3,2-h]-isoquinoline-2,3-dione-3-oxim;
8-methyl-5-phenyl-6-7-8-9-tetrahydro-1H-pyrrolo-[3,2-h]-isoquinoline-2,3-dione-3-oxim;
8-methyl-5-phenyl-6-7-8-9-tetrahydro-1H-pyrrolo-[3,2-h]-isoquinoline-2,3-dione-3-methyloxim;
5-phenyl-6-7-8-9-tetrahydro-1H-pyrrolo-[3,2-h]-isoquinoline-2,3-dione-3-oxim;
5-(4-chlorophenyl)-8-methyl-6-7-8-9-tetrahydro-1-H-pyrrolo-[3,2-h]-isoquinoline-2,3-dione-3-oxim;
5-(2-naphthyl)-8-methyl-6-7-8-9-tetrahydro-1-H-pyrrolo-[3,2-h]-isoquinoline-2,3-dione-3-oxim;
8-methyl-5-phenyl-6,7,8,9-tetrahydro-1H-pyrrolo-[3,2-h]-isoquinoline-2,3-dione-3-acetyloxim;
8-methyl-5-(4-(N,N-dimethylsulfamoyl)-phenyl)-6,7,8,9-tetrahydro-1H-pyrrolo-[3,2-h]-isoquinoline-2,3-dione-3-oxime;
8-benzyl-5-Phenyl-6-7-8-9-tetrahydro-1H-pyrrolo-[3,2-h]-isoquinoline-2,3-dione-3-oxim;
8-methoxycarbonylmethyl-5-phenyl-6,7,8,9-tetrahydro-1H-pyrrolo-[3,2-h]-isoquinoline-2,3-dione-3-oxime;
8-(2-propynyl)-5-phenyl-6,7,8,9-tetrahydro-1H-pyrrolo-[3,2-h]-isoquinoline-2,3-dione-3-oxime; or
8-cyclopropylmethyl-5-phenyl-6,7,8,9-tetrahydro-1H-pyrrolo-[3,2-h]-isoquinoline-2,3-dione-3-oxime;
or a pharmaceutically acceptable salt thereof.
In a fourth preferred embodiment, the compound for use according to the present invention is represented by the general Formula IV 
or a pharmaceutically acceptable salt thereof,
wherein R1, R6, R12, X, Y and n have the meanings set forth above.
In a fifth preferred embodiment, the compound for use according to the present invention is represented by the general Formula V 
or a pharmaceutically acceptable salt thereof,
wherein R1, R6, R12 and X have the meanings set forth above.
In a sixth preferred embodiment, the compound for use according to the present invention is represented by the general Formula VI 
or a pharmaceutically acceptable salt thereof,
wherein R1, R6, R12, X, Y and n have the meanings set forth above.
In a seventh preferred embodiment, the compound for use according to the present invention is represented by the general Formula VII 
or a pharmaceutically acceptable salt thereof,
wherein R1, R6, R12 and X have the meanings set forth above.
In a more preferred embodiment, the compound represented by Formula VII is
7-ethyl-5-phenyl-6,7,8,9-tetrahydro-1H-pyrrolo-[3,2-f]-isoquinoline-2,3-dione-3-oxim; or
5-phenyl-7-methyl-6-7-8-9-tetrahydro-1-methyl-pyrrolo-[3,2-f]-isoquinoline-2,3-dione-3-oxim;
or a pharmaceutically acceptable salt thereof.
In an eight preferred embodiment, the compound for use according to the present invention is represented by the general Formula VIII 
or a pharmaceutically acceptable salt thereof,
wherein R1, R6, R12, X, Y and n have the meanings set forth above.
In a ninth preferred embodiment, the compound for use according to the present invention is represented by the general Formula IX 
or a pharmaceutically acceptable salt thereof,
wherein R1, R6, R12 and X have the meanings set forth above.
In a more preferred embodiment, the compound represented by Formula IX is
7-methyl-5-phenyl-1-6-7-8-tetrahydrobenzo-[2,1-b:3,4-c]-dipyrrole-2,3-dione-3-oxime;
7-methyl-5-(1-naphthyl)-1-6-7-8-tetrahydrobenzo-[2,1-b:3,4-c]-dipyrrole-2,3-dione-3-oxime; or
7-ethyl-5-phenyl-1-6-7-8-tetrahydrobenzo-[2,1-b:3,4-c]-dipyrrole-2,3-dione-3-oxime;
or a pharmaceutically acceptable salt thereof.
In a tenth preferred embodiment, the compound for use according to the present invention is represented by the general Formula X 
or a pharmaceutically acceptable salt thereof,
wherein R1, R6, X, Y and n have the meanings set forth above.
In an eleventh preferred embodiment, the compound for use according to the present invention is represented by the general Formula XI 
or a pharmaceutically acceptable salt thereof,
wherein R1, R6, and X have the meanings set forth above.
In a more preferred embodiment, the compound represented by Formula XI is
5-phenyl-6-7-8-9-tetrahydro-1-H-pyrrolo-[3,2-h]-naphthalene-2,3-dione-3-oxim; or
5-(4-chlorophenyl)-6-7-8-9-tetrahydro-1-H-pyrrolo-[3,2-h]-naphthalene-2,3-dione-3-oxim;
or a pharmaceutically acceptable salt thereof.
In a twelfth preferred embodiment, the compound for use according to the present invention is represented by the general Formula XII 
or a pharmaceutically acceptable salt thereof,
wherein R1, R6, X, Y and n have the meanings set forth above.
In a thirteenth preferred embodiment, the compound for use according to the present invention is represented by the general Formula XIII 
or a pharmaceutically acceptable salt thereof,
wherein R1, R6, and X have the meanings set forth above.
In a fourteenth preferred embodiment, the compound for use according to the present invention is
N-amidino-3,5-diamino-6-chloropyrazine-2-carboxamide (Amiloride).
Definition of Substituents
In the context of this invention halogen represents a fluorine, a chlorine, a bromine or an iodine atom. Thus, a trihalogenmethyl group represents e.g. a trifluoromethyl group and a trichloromethyl group.
In the context of this invention an alkyl group designates a univalent saturated, straight or branched hydrocarbon chain. The hydrocarbon chain preferably contain of from one to eighteen carbon atoms (C1-18-alkyl), more preferred of from one to six carbon atoms (C1-6-alkyl; lower alkyl), including pentyl, isopentyl, neopentyl, tertiary pentyl, hexyl and isohexyl. In a preferred embodiment alkyl represents a C1-4-alkyl group, including butyl, isobutyl, secondary butyl, and tertiary butyl. In another preferred embodiment of this invention alkyl represents a C1-3-alkyl group, which may in particular be methyl, ethyl, propyl or isopropyl.
In the context of this invention an alkenyl group designates a carbon chain containing one or more double bonds, including di-enes, tri-enes and poly-enes. In a preferred embodiment the alkenyl group of the invention comprises of from two to eight carbon atoms (C2-8-alkenyl), more preferred of from two to six carbon atoms (C2-6-alkenyl), including at least one double bond. In a most preferred embodiment the alkenyl group of the invention is ethenyl; 1- or 2-propenyl; 1-, 2- or 3-butenyl, or 1,3-butenyl; 1-, 2-, 3-, 4- or 5-hexenyl, or 1,3-hexenyl, or 1,3,5-hexenyl; 1-, 2-, 3-, 4-, 5-, 6-, or 7-octenyl, or 1,3-octenyl, or 1,3,5-octenyl, or 1,3,5,7-octenyl.
In the context of this invention an alkynyl group designates a carbon chain containing one or more triple bonds, including di-ynes, tri-ynes and poly-ynes. In a preferred embodiment the alkynyl group of the invention comprises of from two to eight carbon atoms (C2-8-alkynyl), more preferred of from two to six carbon atoms (C2-6-alkynyl), including at least one triple bond. In its most preferred embodiment the alkynyl group of the invention is ethynyl; 1-, or 2-propynyl; 1-, 2-, or 3-butynyl, or 1,3-butynyl; 1-, 2-, 3-, 4-pentynyl, or 1,3-pentynyl; 1-, 2-, 3-, 4-, or 5-henynyl, or 1,3,5-hexynyl or 1,3,5-hexynyl; 1-, 2-, 3-, 4-, 5- or 6-heptynyl, or 1,3-heptynyl, or 1,3,5-heptynyl; 1-, 2-, 3-, 4-, 5-, 6- or 7-octynyl, or 1,3-octynyl, or 1,3,5-octynyl, or 1,3,5,7-octynyl.
In the context of this invention a cycloalkyl group designates a cyclic alkyl group, preferably containing of from three to seven carbon atoms (C3-7-cycloalkyl), including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
In the context of this invention a cycloalkyl-alkyl group designates a cycloalkyl group as defined above, which cycloalkyl group is substituted on an alkyl group as also defined above. Examples of preferred cycloalkyl-alkyl groups of the invention include cyclopropylmethyl and cyclopropylethyl.
In the context of this invention an alkoxy group designates an xe2x80x9calkyl-Oxe2x80x94xe2x80x9d group, wherein alkyl is as defined above.
In the context of this invention an alkoxy-alkyl group designates an xe2x80x9calkyl-O-alkyl-xe2x80x9d group, wherein alkyl is as defined above.
In the context of this invention an acyl group designates a carboxy group (xe2x80x94COOH) or an alkyl-carbonyl group (alkyl-COxe2x80x94), wherein alkyl is as defined above. Examples of preferred acyl groups of the invention include carboxy, acetyl, and propionyl.
In the context of this invention an alkoxy-carbonyl-alkyl group designates an xe2x80x9calkyl-Oxe2x80x94CO-alkylxe2x80x9d group, wherein alkyl is as defined above.
In the context of this invention an alkyl-carbonyl-oxy-alkyl group designates an xe2x80x9calkyl-COxe2x80x94O-alkyl-xe2x80x9d group, wherein alkyl is as defined above.
In the context of this invention an amino group may be a primary (xe2x80x94NH2), secondary (xe2x80x94NH-alkyl), or tertiary (xe2x80x94N(alkyl)2) amino group, i.e. it may be substituted once or twice with an alkyl group as defined above.
Pharmaceutically Acceptable Salts
The chemical compound of the invention may be provided in any form suitable for the intended administration. Suitable forms include pharmaceutically (i.e. physiologically) acceptable salts, and pre- or prodrug forms of the chemical compound of the invention.
Examples of pharmaceutically acceptable addition salts include, without limitation, the non-toxic inorganic and organic acid addition salts such as the hydrochloride derived from hydrochloric acid, the hydrobromide derived from hydrobromic acid, the nitrate derived from nitric acid, the perchlorate derived from perchloric acid, the phosphate derived from phosphoric acid, the sulphate derived from sulphuric acid, the formate derived from formic acid, the acetate derived from acetic acid, the aconate derived from aconitic acid, the ascorbate derived from ascorbic acid, the benzenesulphonate derived from benzensulphonic acid, the benzoate derived from benzoic acid, the cinnamate derived from cinnamic acid, the citrate derived from citric acid, the embonate derived from embonic acid, the enantate derived from enanthic acid, the fumarate derived from fumaric acid, the glutamate derived from glutamic acid, the glycolate derived from glycolic acid, the lactate derived from lactic acid, the maleate derived from maleic acid, the malonate derived from malonic acid, the mandelate derived from mandelic acid, the methanesulphonate derived from methane sulphonic acid, the naphthalene-2-sulphonate derived from naphtalene-2-sulphonic acid, the phthalate derived from phthalic acid, the salicylate derived from salicylic acid, the sorbate derived from sorbic acid, the stearate derived from stearic acid, the succinate derived from succinic acid, the tartrate derived from tartaric acid, the toluene-p-sulphonate derived from p-toluene sulphonic acid, and the like. Such salts may be formed by procedures well known and described in the art.
Other acids such as oxalic acid, which may not be considered pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining a chemical compound of the invention and its pharmaceutically acceptable acid addition salt.
Metal salts of a chemical compound of the invention includes alkali metal salts, such as the sodium salt of a chemical compound of the invention containing a carboxy group.
In the context of this invention the xe2x80x9conium saltsxe2x80x9d of N-containing compounds are also contemplated as pharmaceutically acceptable salts. Preferred xe2x80x9conium saltsxe2x80x9d include the alkyl-onium salts, the cycloalkyl-onium salts, and the cycloalkylalkyl-onium salts.
The chemical compound of the invention may be provided in dissoluble or indissoluble forms together with a pharmaceutically acceptable solvents such as water, ethanol, and the like. Dissoluble forms may also include hydrated forms such as the monohydrate, the dihydrate, the hemihydrate, the trihydrate, the tetrahydrate, and the like. In general, the dissoluble forms are considered equivalent to indissoluble forms for the purposes of this invention.
Steric Isomers
The chemical compounds of the present invention may exist in (+) and (xe2x88x92) forms as well as in racemic forms. The racemates of these isomers and the individual isomers themselves are within the scope of the present invention.
Racemic forms can be resolved into the optical antipodes by known methods and techniques. One way of separating the diastereomeric salts is by use of an optically active acid, and liberating the optically active amine compound by treatment with a base. Another method for resolving racemates into the optical antipodes is based upon chromatography on an optical active matrix. Racemic compounds of the present invention can thus be resolved into their optical antipodes, e.g., by fractional crystallisation of d- or I-(tartrates, mandelates, or camphorsulphonate) salts for example.
The chemical compounds of the present invention may also be resolved by the formation of diastereomeric amides by reaction of the chemical compounds of the present invention with an optically active activated carboxylic acid such as that derived from (+) or (xe2x88x92) phenylalanine, (+) or (xe2x88x92) phenylglycine, (+) or (xe2x88x92) camphanic acid or by the formation of diastereomeric carbamates by reaction of the chemical compound of the present invention with an optically active chloroformate or the like.
Additional methods for the resolving the optical isomers are known in the art. Such methods include those described by Jaques J, Collet A, and Wilen S in xe2x80x9cEnantiomers, Racemates, and Resolutionsxe2x80x9d, John Wiley and Sons, New York (1981).
Optical active compounds can also be prepared from optical active starting materials.
Moreover, some of the chemical compounds of the invention being oximes, may thus exist in two forms, syn- and anti-form (Z- and E-form), depending on the arrangement of the substituents around the xe2x80x94Cxe2x95x90Nxe2x80x94 double bond. A chemical compound of the present invention may thus be the syn- or the anti-form (Z- and E-form), or it may be a mixture hereof.
Methods of Preparation
The chemical compounds of the invention may be prepared by conventional methods for chemical synthesis, e.g. those described in WO 94/26747, WO 96108494 and WO 96108495, and in the working examples. The starting materials for the processes described in the present application are known or may readily be prepared by conventional methods from commercially available chemicals.
Also one compound of the invention can be converted to another compound of the invention using conventional methods.
The end products of the reactions described herein may be isolated by conventional techniques, e.g. by extraction, crystallisation, distillation, chromatography, etc.
Biological Activity
The presence of ASIC in central neurons is of particular interest because tissue acidosis is a well established feature of cerebral ischaemia. While the acidic pH in general has been regarded as neuro-protective, due to proton inhibition of NMDA receptors, it may also have adverse effects by reason of activation of acid sensing ion channels contributing to membrane depolarisation, subsequent Ca2+ accumulation and neuro-degeneration. In this respect it seems justified that the administration of ASIC antagonising compounds provide protection to individuals against injury arising from a drop in extracellular pH.
Diseases and disorders contemplated according to the present invention include in particular diseases of the central nervous system (CNS). In particular, the invention relates to combating diseases associated with reduced blood flow to the brain and other CNS tissue and with instances of a temporary break in blood supply to the brain or to other CNS tissue. Examples include ischaemic diseases, anoxic episodes, and injury to the brain and other parts of the CNS caused by trauma or other injury, for example a blow to the head, or spinal injury. In such reduced blood flow episodes, or episodes where there is a temporary break in blood supply, oxygen supply to the brain is reduced or interrupted.
Diseases and disorders contemplated according to the present invention also include cerebrovascular disorders such as cerebral ischaemia or cerebral infarction resulting from a range of conditions, such as tromboembolic or haemorrhagic stroke, cerebral vasospasm, hypoglycaemia, cardiac arrest, status epilepticus, perinatal asphyxia, anoxia such as from near-drowning, pulmonary surgery and cerebral trauma as well as lathyrism, Alzheimer""s disease, and Huntington""s disease. The method can be used in the treatment or prevention of traumatic brain injury, in particular ischaemic, hypoxic or anoxic brain damage, spinal cord injury, tissue ischaemia and reperfusion injury in a mammal at risk for such damage.
The brain damage may follow cerebral ischaemia, either global or focal, or be caused by cardiac arrest, or may follow high risk surgery such as cardiac surgery. It may also follow or be caused by stroke, neonatal hypoxia, hypoxia caused by compromised lung function, neonatal anoxia, anoxia caused by compromised lung function, cerebral trauma, secondary regional ischaemia induced by brain oedema, increased intercranial pressure, open brain surgery, endarterectomy, surgical interventions involving temporary, artificially sustained arrest of cardiopulmonary functions resulting in impairment of cerebral blood flow, and emergency treatment involving cardiopulmonary resuscitation (CPR).
As used herein, reperfusion injury refers to the cellular changes and tissue damage seen after a period of total ischaemia followed by reperfusion. Extremity re-plantation, organ transplantation, free flap tissue reconstruction and even myocardial infarction and stroke are all clinical examples of interval tissue ischaemia which can lead to tissue loss due to reperfusion injury after blood flow is re-established. Tissue reperfusion injury, seen in its full clinical extent as the no-reflow phenomenon, appears as inflammatory response to reperfusion resulting in the ultimate death of the tissue.
Thus the chemical compounds of the invention are found to be particularly useful in acute treatment of ischaemic stroke, in treatment of brain damage following global cerebral ischaemia, or for prevention of brain damage following high risk surgery.
In many instances of brain ischaemia, treatment is not available to the patient for several, e.g. up to 6 hours, in stroke patients typically 3 to 6 hours, after the ischaemic injury. Such a delay places great demands on any therapeutic regime designed to mitigate ischaemic brain injury.
When administered post-ischaemically it is advisable that the chemical compounds of the invention be administered within one day of the ischaemic insult. Although the neuro-protective agent used in the invention may be administered as late as 14 hours after brain reperfusion, the treatment should preferably be carried out within 12 hours of ischaemic alleviation or reperfusion. Preferably, the treatment should occur within 6 hours of alleviation of ischaemia. Yet more preferred is the administration of the chemical compounds used in the invention within 3 hours of alleviation of ischaemia.
Pharmaceutical Compositions
Thus viewed from one aspect, the invention provides ASIC antagonising compounds for use in the manufacture of a pharmaceutical composition for treatment or alleviation of diseases and disorders associated with or mediated by a drop in extracellular pH.
In a preferred embodiment of this aspect, pharmaceutical compositions for treatment or alleviation of diseases and disorders associated with or mediated by a drop in extracellular pH, which pharmaceutical compositions comprise a pharmaceutically effective amount of an ASIC antagonising compound together with a pharmaceutically effective amount of an AMPA receptor antagonising compound.
While a chemical compound of the invention for use in therapy may be administered in the form of the raw chemical compound, it is preferred to introduce the active ingredient, optionally in the form of a physiologically acceptable salt, in a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffers, diluents, and/or other customary pharmaceutical auxiliaries.
In a preferred embodiment, the invention provides pharmaceutical compositions comprising an ASIC antagonising compound or a pharmaceutically acceptable salt or derivative thereof, together with one or more pharmaceutically acceptable carriers therefor, and, optionally, other therapeutic and/or prophylactic ingredients, know and used in the art. The carrier(s) must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof.
The pharmaceutical composition of the invention may be administered by any convenient route which suite the desired therapy. Preferred routes of administration include oral administration, in particular in tablet, in capsule, in drage, in powder, or in liquid form, and parenteral administration, in particular cutaneous, subcutaneous, intramuscular, or intravenous injection. The pharmaceutical composition may be prepared by the skilled person using standard and conventional techniques appropriate to the desired formulation. When desired, compositions adapted to give sustained release of the active ingredient may be employed.
Further details on techniques for formulation and administration may be found in the latest edition of Remington""s Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
The actual dosage depend on the nature and severity of the disease being treated, and is within the discretion of the physician, and may be varied by titration of the dosage to the particular circumstances of this invention to produce the desired therapeutic effect. However, it is presently contemplated that pharmaceutical compositions containing of from about 0.1 to about 500 mg of active ingredient per individual dose, preferably of from about 1 to about 100 mg, most preferred of from about 1 to about 10 mg, are suitable for therapeutic treatments.
The active ingredient may be administered in one or several doses per day. A satisfactory result can, in certain instances, be obtained at a dosage as low as 0.1 xcexcg/kg i.v. and 1 xcexcg/kg p.o. The upper limit of the dosage range is presently considered to be about 10 mg/kg i.v. and 100 mg/kg p.o. Preferred ranges are from about 0.1 xcexcg/kg to about 10 mg/kg/day i.v., and from about 1 xcexcg/kg to about 100 mg/kg/day p.o.
Viewed from another aspect, the invention provides a method for treatment, prevention or alleviation of a disease or a disorder or a condition of a living animal body, including a human, which disease or a disorder or a condition is associated with, or mediated by a drop in extracellular pH, said method comprising administering to a subject a pharmaceutically effective amount of a proton-gated cation channel inhibiting compound.
In a preferred embodiment, the disease, disorder or condition is associated with reduced blood flow to the brain and other CNS tissue, or associated with instances of a temporary break in blood supply to the brain or to other CNS tissue.
In another preferred embodiment, the disease, disorder or condition is an ischaemic disease, an anoxic episode, an injury to the brain and other parts of the CNS caused by trauma or other injury, a blow to the head, or a spinal injury, a tromboembolic or haemorrhagic stroke, a cerebral vasospasm, hypoglycaemia, cardiac arrest, status epilepticus, perinatal asphyxia, anoxia, cerebral trauma, lathyrism, Alzheimer""s disease, and Huntington""s disease, cerebral ischaemia or cerebral infarction, ischaemic, hypoxic or anoxic brain damage, spinal cord injury, tissue ischaemia and reperfusion injury in a mammal at risk for such damage.
In a third preferred embodiment of this aspect, the method of the invention comprises co-administration of an ASIC antagonising compound and a pharmaceutically effective amount of an AMPA receptor antagonising compound.
In a more preferred embodiment, the AMPA receptor antagonising compound is 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo-(f)-quinoxaline (NBQX), 6,7-dinitroquinoxaline-2,3-dione, or 6-cyano-7-nitroquinoxaline-2,3-dione, or a pharmaceutically acceptable salt thereof.
It is at present contemplated that suitable dosage ranges are 0.1 to 1000 milligrams daily, 10-500 milligrams daily, and especially 30-100 milligrams daily, dependent as usual upon the exact mode of administration, form in which administered, the indication toward which the administration is directed, the subject involved and the body weight of the subject involved, and further the preference and experience of the physician or veterinarian in charge.
AMPA receptor antagonising compounds are well known and described in the literature.
Preferred examples of AMPA receptor antagonising compounds include those described in e.g. EP 451626, EP 432648, EP 503349, EP 522494, WO 93/05043, EP 633262, WO 94/26747, EP 629615, EP 667340, WO 96/08494, WO 96/08495, WO 96/08494 and WO 98/14447, and the quinoxaline- or quinoxalinedione derivatives, in particular 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo-(f)-quinoxaline (NBQX), and 6,7-dinitroquinoxaline-2,3-dione or 6-cyano-7-nitroquinoxaline-2,3-dione.
The actual dosage depend on the nature and severity of the disease being treated, and is within the discretion of the physician, and may be varied by titration of the dosage to the particular circumstances of this invention to produce the desired therapeutic effect. However, it is presently contemplated that pharmaceutical compositions containing of from about 0.1 to about 500 mg of active ingredient per individual dose, preferably of from about 1 to about 100 mg, most preferred of from about 1 to about 100 mg, are suitable for therapeutic treatments.
The active ingredient may be administered in one or several doses per day. A satisfactory result can, in certain instances, be obtained at a dosage as low as 0.1 xcexcg/kg i.v. and 1 xcexcg/kg p.o. The upper limit of the dosage range is presently considered to be about 10 mg/kg i.v. and 100 mg/kg p.o. Preferred ranges are from about 0.1 xcexcg/kg to about 10 mg/kg/day i.v., and from about 1 xcexcg/kg to about 100 mg/kg/day p.o.